TY - JOUR
T1 - Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32
AU - Sunayama, J.
AU - Ando, Y.
AU - Itoh, N.
AU - Tomiyama, A.
AU - Sakurada, K.
AU - Sugiyama, A.
AU - Kang, D.
AU - Tashiro, F.
AU - Gotoh, Y.
AU - Kuchino, Y.
AU - Kitanaka, C.
N1 - Funding Information:
We thank Drs. Naohiro Inohara and Yoshihide Tsujimoto for the generous gift of Hrk and Bcl-xL expression vectors, respectively, Dr. Jun Yokota for critical reading of the paper, and Tomo Yasuda for technical assistance. We also thank members of the Gotoh laboratory for encouragement and helpful discussions. This work was supported by Grants-in-Aid for Scientific Research and for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Grant-in-Aid for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor, and Welfare of Japan.
Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2004/7
Y1 - 2004/7
N2 - Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.
AB - Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.
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U2 - 10.1038/sj.cdd.4401418
DO - 10.1038/sj.cdd.4401418
M3 - Article
C2 - 15031724
AN - SCOPUS:3543075659
VL - 11
SP - 771
EP - 781
JO - Cell Death and Differentiation
JF - Cell Death and Differentiation
SN - 1350-9047
IS - 7
ER -